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77Journal of Cell and Molecular Biology 4: 77-86, 2005.Haliç University, Printed in Turkey.Molecular farming in plants: An approach of agricultural biotechnologyKunka Kamenarova1*, Nabil Abumhadi1, Kostadin Gecheff2and Atanas Atanassov11AgroBioInstitute, 8, Dragan Tzankov Blvd., 1164 Sofia, Bulgaria2Bulgarian Academy of Science, 1113Sofia, Bulgaria (*author for correspondence)Received 17 March 2005; Accepted 25 April 2005AbstractMolecular farming is defined as the production of proteins or other metabolites valuable to medicine or industry inplants traditionally used in an agricultural setting. Crop plants can synthesize a wide variety of proteins that are freeof mammalian toxins and pathogens. Crop plants produce large amounts of biomass at low cost and require limitedfacilities. Since plants have long been used as a source of medicinal compounds, molecular farming represents anovel source of molecular medicines, such as plasma proteins, enzymes, growth factors, vaccines and recombinantantibodies, whose medical applications are understood at a molecular level. Bio-pharming promises more plentifuland cheaper supplies of pharmaceutical drugs, including vaccines for infectious diseases and therapeutic proteins fortreatment of such things as cancer and heart disease. “Plant-made pharmaceuticals” (PMPs) are produced bygenetically engineering plants to produce specific compounds, generally proteins, which are extracted and purifiedafter harvest. As used here, the terms molecular farming and PMP do not include naturally occurring plant productsor nutritionally enhanced foods.Key words:molecular farming, plant-made pharmaceuticals, recombinant proteins, secretion pathwaysBitkilerde moleküler tar›m: Tar›msal biyoteknolojiye yaklaﬂ›mÖzetMoleküler tar›m, ilaç veya endüstri aç›s›ndan de¤erli geleneksel olarak kullan›lan proteinlerin veya di¤ermetabolitlerin üretimi olarak tan›mlan›r. Bitkiler memeli toksinleri ve patojenleri içermeyen çok çeﬂitli proteinlerisentez edebilir. Bitkiler düﬂük fiyata mal olan ve s›n›rl› olanaklara gerek duyan fazla miktarda biyomas üretebilir.Bitkiler çok uzun süredir t›bbi bileﬂenlerin kayna¤› olarak kullan›ld›¤›ndan, moleküler tar›m, t›bbi kullan›mlar›moleküler düzeyde anlaﬂ›lm›ﬂ olan plazma proteinleri, enzimler, büyüme faktörleri, aﬂ›lar ve rekombinant antikorlargibi moleküler ilaçlar için yeni kaynaklard›r. Biyo–tar›m, enfeksiyon hastal›klar için gerekli aﬂ›lar, kanser ve kalphastal›klar› için kullan›lan teröpatik proteinleri içeren bitkisel ilaçlar›n daha bol ve daha ucuza elde edilmesi içinumut vericidir. “Bitki yap›m› farmasotikler” (PMPS) genetik mühendisli¤i ile bitkilerden spesifik bileﬂikler,genellikle hasat sonras› ekstre edilip saflaﬂt›r›lan proteinlerin üretilmesi için kullan›lmaktad›r. Burada da kullan›ld›¤›gibi moleküler tar›m ve PMP do¤al meydana gelen bitkisel ürünleri veya besleyici de¤eri artt›r›lm›ﬂ besinleriiçermemektedir.Anahtar sözcükler:Moleküler tar›m, bitki–yap›m› ilaçlar, rekombinant proteinler, salg› yolaklar›IntroductionManufacturing pharmaceutical products in crops hasbeen one of the promised benefits of plant geneticengineering for the past 20 years. The using ofbiotechnology, sometimes known as “pharming,”“bio-pharming,” or “molecular farming,” has migratedfrom speculation to the testing phase in fields andgreenhouses across the country.While short peptide chains (containing fewer than30 amino acids) can be synthesized chemically, largerproteins are best produced by living cells. The DNAthat encodes the instructions for producing the desiredprotein is inserted into cells, and as the cells grow theysynthesize the protein, which is subsequentlyharvested and purified. Plants have provided humanswith useful molecules for many centuries, but only inthe past 20 years it has became possible to use plantsfor the production of specific heterologous proteins.The use of transgenic higher plants to produce foreignproteins with economic value was being realized(Kusnadi et al., 1998). The first pharmaceuticallyrelevant protein made in plants was human growthhormone, which was expressed in transgenic tobaccoin 1986 (Barta, 1986). During the period 1986- 1999many therapeutics produced in plants were reportedfor first time: human antibodies (During, 1988);secretory antibodies (Hiatt et al., 1989); egg proteinswith important properties - avidin (Hood et al., 1997)and aprotinin, one of the first molecularly farmedpharmaceutical proteins produced in plants (Zhong etal., 1999).Although transgenic animals, bacteria and fungiare also utilized for the production of proteins, highesteconomic benefit will likely be achieved with plants(Horn et al., 2004). Many protein-based drugs arecurrently produced in sterile fermentation facilities bygenetically engineered microorganisms or mammaliancell cultures in stainless steel tanks. Another methodfor obtaining biopharmaceuticals is to extract themfrom animal and human tissues (e.g., insulin from pigand cow pancreas, or blood proteins from humanblood (Freese, 2002). However, these are high-costprocedures that carry the risk of transmittinginfectious diseases to humans. Due to advances inplant genetic engineering over the past two decades,plants can now be modified to produce a wide range ofproteins. It is hoped this will result in therapeuticproducts at a price significantly cheaper than thoseobtained by the currently applied methods (Table 1).The idea for using plants to produce humanproteins was initially met with great skepticism.However, plants offer a unique combination ofadvantages over traditional microbial and animalexpression systems. Molecular farming in plantsbegan in earnest in 1989 with the remarkabledemonstration that functional recombinant antibodiescould be expressed in tobacco (Hiatt et al., 1989).Before this result was published, there was littlesupport for the idea that plants could be used toproduce therapeutic proteins. Since then, it has beenshown that transgenic plants are extremely versatileand they have been used to produce a wide range ofpharmaceutical proteins (Schillberg et al., 2003).According to Horn (Horn et al., 2004) the advantagesof using higher plants for the purpose of proteinproduction include: (1) significantly lower productioncosts than with transgenic animals, fermentation or78 Kunka Kamenarova et al.Table 1: Comparison of production systems for recombinant human pharmaceutical proteins (Ma et al., 2003).System Overall Production Scale up Product Glycosylation Contamination Storagecost timescale capacity quality risk costBacteria Low Short High Low None Endotoxins ModerateYeast Medium Medium High Medium Incorrect Low risk ModerateTransgenic Viruses,animals High Very long Low Very high Correct Oncogenic NA ExpensivePlant cell Minorcultures Medium Medium Medium High differences Low risk ModerateTransgenic Very Very Minorplants Low Long High High differences Low risk Inexpensivebioreactors; (2) infrastructure and expertise alreadyexists for the planting, harvesting and processing ofplant material; (3) plants do not contain known humanpathogens (such as virions, etc.) that couldcontaminate the final product; (4) higher plantsgenerally synthesize proteins from eukaryotes withcorrect folding, glycosylation, and activity; and (5)plant cells can direct proteins to environments thatreduce degradation and therefore increase stability.Recombinant proteins expressed in plantsUntil recently, pharmaceuticals used for the treatmentof diseases have been based largely on the productionof relatively small organic molecules, chemically ormicrobially synthesized. Presently, attention isfocused on larger and more complex proteinmolecules as therapeutic agents. Examples of proteinsthat have been produced in plants are listed in table 2.Horn (Horn et al., 2004) categorizes proteinscurrently being produced in plants for molecularfarming purposes into four broad areas: (1) parentaltherapeutics and pharmaceutical intermediates, (2)industrial proteins (e.g., enzymes), (3) monoclonalantibodies (MAbs), and (4) antigens for ediblevaccines.The group of parental therapeutics andpharmaceutical intermediatesIncludes all proteins used directly as pharmaceuticalsalong with those proteins used in the making ofpharmaceuticals. The list of such proteins is long, evergrowing, and includes such products as thrombin andcollagen (therapeutics), and trypsin and aprotinin(intermediates).Industrial proteinsThis group includes hydrolases, encompassing bothglycosidases and proteases. Enzymes involved inbiomass conversion for producing ethanol arecandidates for molecular farming. All of theseproducts are usually characterized by the fact that theyare used in very large quantities and must therefore beproduced very inexpensively (Hood et al., 1999).Recombinant monoclonal antibodiesThis group includes all antibody forms (IgA, IgG,IgM, secretory IgA, etc.) and antibody fragments (Fv).They can be produced in plants in both glycosylatedand nonglycosylated forms.Plants are an alternative expression system toanimals for the molecular farming of antibodies(Schillberg et al., 2003). The production of antibodiesin plants represents a special challenge because themolecules must fold and assemble correctly torecognize their cognate antigens. Typical serumantibodies are tetramers of two identical heavy chainsand two identical light chains; however, there are morecomplex forms, such as secretory antibodies, whichare dimers of the typical serum antibody and includetwo extra polypeptide chains. Two different cell typesare required to assemble such antibodies in mammals,but plants that express four different transgenes canassemble these antibodies in a single cell (Ma et al.,2003).Transgenic plants have been used for theproduction of antibodies directed against dental caries,rheumatoid arthritis, cholera, E. coli diarrhea, malaria,certain cancers, Norwalk virus, HIV, rhinovirus,influenza, hepatitis B virus, and herpes simplex virus(Thomas et al., 2002). Some of these havedemonstrated preventative or therapeutic value and arecurrently in clinical trials.Antigens for edible vaccinesPlant-derived vaccines have been produced againstVibrio cholerae, enterotoxigenic E. coli, hepatitis Bvirus, Norwalk virus, rabies virus, humancytomegalovirus, rotavirus and respiratory syncytialvirus F (Thomas et al., 2002). Antigens specific to anindividual patient’s tumor are expressed in tobacco,harvested, purified, and administered to the patient.This entire process can take as little as 4 weeks,compared to 9 months for the same process usingmammalian cell culture.Many of these plant-derived antigens were purifiedand used as injectable vaccines, but oral delivery ofthese vaccines within foods has also been successful.In some cases, protection has actually been better withthe edible vaccine than with the commerciallyavailable vaccine (Lamphear et al. 2004). In this wayit could be overcome the need for injections and sterileneedles and do not require refrigeration. Ediblevaccines are being tested in potatoes, tomatoes,bananas, and carrots. Potatoes are usually cooked forconsumption, which may inactivate the vaccine. ShortMolecular farming in plants 7980 Kunka Kamenarova et al.Table 2: Important pharmaceutical proteins that have been produced in plants (Thomas et al., 2002; Ma et al., 2003).Protein Host plant system Comments/ Medical applicationHuman biopharmaceuticalsGrowth hormone Tobacco, sunflower First human protein expressed in plants; initially expressed as fusionprotein with nos gene in transgenic tobacco; later the first humanprotein expressed in chloroplasts, with expression levels ~7% of totalleaf proteinHuman serum albumin Tobacco, potato First full size native human protein expressed in plants; low expressionlevels in transgenics (0.1% of total soluble protein) but high levels(11% of total leaf protein) in transformed chloroplasts/ Liver cirrhosis,burns, surgeryα-interferon Rice, turnip First human pharmaceutical protein produced in riceErythropoietin Tobacco First human protein produced in tobacco suspension cells/ AnemiaHuman-secretedalkaline phosphatase Tobacco Produced by secretion from roots and leavesAprotinin Maize Human pharmaceutical protein produced in maizeCollagen Tobacco First human structural-protein polymer produced in plant; correctmodification achieved by co-transformation with modification enzymeα1-antitrypsin Rice First use of rice suspension cells for molecular farmingLactoferrin Rice, tomato Antimicrobal activityProtein C Tobacco AnticoagulantHirudin Canola Thrombin inhibitorGranulocyte-macrophagecolony-stimulating factor Tobacco NeutropeniaEnkephalins Arabidopsis Antihyperanalgesic by opiate activityEpidermal growth Tobacco Wound repair and control of cell proliferationRecombinant antibodiesImmunoglobulin G1 Tobacco First antibody expressed in plants; full length serum IgG produced bycrossing plants that expressed heavy and light chainsImmunoglobulin M Tobacco First IgM expressed in plants and protein targeted to chloroplasts foraccumulationSecretoryimmunoglobulin A Tobacco First secretory antibody expressed in plants by sequential crossing offour lines carrying individual components; at present the mostadvanced plant-derived pharmaceutical proteinImmunoglobulin G(herpes simplex virus) Soybean First pharmaceutical protein produced in soybeanHepatitis B virusenvelope protein Tobacco First vaccine candidate expressed in plants; third plant-derivedvaccine to reach clinical trials stageRabies virus glycoprotein Tomato First example of an ‘edible vaccine’ expressed in edible plant tissueEscherichia coli heat-labileenterotoxin Tobacco, potato First plant vaccine to reach clinical trials stageDiabetes autoantigen Tobacco, potato First plant-derived vaccine for an autoimmune diseaseCholera toxin B subunit Tobacco, potato First vaccine candidate expressed in chloroplastsCholera toxin B and A2subunits, rotavirusenterotoxin Potato First example of oral feeding inducing protection in an animalstorage life and length of production cycle may hindervaccine production in tomatoes and bananas. Carrotshave few storage problems and can be eaten raw, andcarrots modified to produce the antigen used inhepatitis B vaccines are currently entering preclinicaltrials.Other proteins of medical relevanceThese include the milk proteins ß-casein, lactoferrinand lysozyme, which could be used to improve childhealth, and protein polymers that could be used insurgery and tissue replacement (Ma et al., 2003).Expression of thioredoxin in foods such as cerealgrains would increase the digestibility of proteins andthereby reduce their allergenicity (Thomas et al.,2002). It has been shown that human collagen can beproduced in transgenic tobacco plants and that theprotein is spontaneously processed and assembled intoits typical triple-helical conformation. The originalplant-derived collagen had a low thermal stabilityowing to the lack of hydroxyproline residues, but thiswas remedied by co-expressing the enzyme proline-4-hydroxylase (Ma et al., 2003). Hood and colleagues(Hood et al., 1997) reported the production of chickenegg white avidin in transgenic corn using an avidingene whose sequence had been optimized forexpression in corn. The resultant avidin had propertiesalmost identical to those of avidin from chicken eggwhite (Horn et al., 2004).Protein expression systemsPlants are genetically enhanced to produce high-valueproteins that are needed for the production of a widerange of therapeutics. The structure and functionalityof a given protein is determined by its sequence ofamino acids, which, in turn, determines its three-dimensional conformation, or structure. Internal bonds(sulfur and hydrogen bonds) among the amino acidsgive the protein its final shape and form. Complexproteins undergo further processing such as theaddition of phosphate groups (phosphorylation) orcarbohydrate molecules (glycosylation), whichmodify the proteins’ functions. Information stored inDNAdirects the protein-synthesizing machinery of thecell to produce the specific proteins required for itsstructure and metabolism.Genetic aspect of producing of PMPsTo achieve specific protein production in plants, theDNAthat encodes the desired protein must be insertedinto the plant cells. This can be done as a stabletransformation when foreign DNAis incorporated intothe genome of the plant. A promoter associated withthe inserted DNAthen directs the cells to produce thedesired protein, often targeting it to accumulate only inspecific tissues such as the seed. Alternatively, a plantvirus can be used to direct expression of a specificprotein without genetically modifying the host plant.The transformation and expression systems used toengineer these proteins in plants affect the stability,Molecular farming in plants 81Table 3: Examples of recombinant proteins targeted to subcellular compartments in transgenic plants.Proteins Host plants Tissue Subcellular Referencesexpression targetsα-Amylase Tobacco Leaves Apoplast Seon et al., 2002Avidin Corn Seeds Apoplast Hood et al., 1997Secretory antibodies Tobacco Leaves Apoplast Hiatt et al., 1989ß-Glucuronidase Brassica Seeds Oil bodies Seon et al., 2002Anti-oxazolone Tobacco Leaves ER Seon et al., 2002Xylanase Brassica Seeds Oil bodies Seon et al., 2002Anti-phytochrome Tobacco Leaves Cytosol Seon et al., 2002Anti- ß-1,4-endoglucanase Potato Roots Cytosol Seon et al., 2002Hirudin Brassica Seeds Oil bodies Seon et al., 2002Vicilin Tobaco,alfalfa Leaves ER Wandelt et al., 1992yield, cost of purification, and quality of the proteinsproduced (Thomas et al., 2002). In addition, themethods used affect the procedures needed to preventthe spread of the engineered traits to other plantsduring their growth in the field.Foreign genes may be inserted, or transformed,into plants via a number of methods. Stabletransformation into the nuclear genome is doneprimarily using Agrobacterium mediatetransformation or particle bombardment methods(Suslow et al., 2002). In each case, the DNA codingfor the protein of interest and an associated promoterto target its expression to a particular tissue ordevelopmental stage is integrated into the genome ofthe plant. Thus, when the plant is propagated, eachplant will transmit this property to its progeny andlarge numbers of plants containing the transferredgene are readily generated. It is also possible to delivergenes into the separate genome of plastids(chloroplasts and mitochondria) in plant cells.Chloroplast transformation has been successful intobacco and potato, and research is being done toexpand to other crops. Because genes in chloroplastgenomes are not transmitted through pollen,recombinant genes are easier to contain, therebyavoiding unwanted escape into the environment. Asecond method of engineering plant protein expressionis transduction, the use of a recombinant plant virus todeliver genes into plant cells. The DNAcoding for thedesired protein is engineered into the genome of aplant virus that will infect a host plant. A crop of thehost plants is grown to the proper stage and is theninoculated with the engineered virus. As the virusreplicates and spreads within the plant, many copies ofthe desired DNA are produced and high levels ofprotein production are achieved in a short time. Alimitation with this system is that the green plantmatter must be processed immediately after harvestand cannot be stored (Thomas et al., 2002).Use of secretion pathways for subcellular targetingTo understand the factors controlling stability andaccumulation of heterologous proteins, it is importantto know where the protein of interest is located withinspecific plant cells or tissues and how this localizationchanges during development and as a result ofenvironmental conditions. Isolation and purification ofthe desired protein may be greatly facilitated bysequestering the protein into a particular cellularcompartment. The secretion pathway in plantsregulates and determines the passage of polypeptidesto tonoplast-derived protein bodies, endoplasmicreticulum (ER)-derived protein bodies or secretioninto the apoplastic space (Table 3). They may undergospecialized folding and post-translational modificationthat requires components of the ER. By including theappropriate signal peptide sequence or fusionresponsible for directing expression and deposition, itis possible to target recombinant proteins to the lumenof the ER, vacuole or other cellular compartments. Asan advantage of this pathway it may be indicated thatthe secretion into one of the cellular compartmentsmay separate the desired protein from proteases likelyto catalyze its breakdown. Secretion has also beenfound to enhance protein stability by facilitatingproper folding. Targeting signals can be used tointentionally retain recombinant proteins withindistinct compartments of the cell to protect them fromproteolytic degradation, preserve their integrity and toincrease their accumulation levels (Seon et al., 2002).In this direction it is now possible to design geneconstructs which contain ER-targeting signal peptide,KDEL, and to increase the level of accumulation offoreign proteins in transgenic plants. The presence ofthe ER-targeting signal led to a greatly enhancedaccumulation of the heterologous protein. Forexample, the gene for the pea seed protein vicilin wasmodified by the addition of a sequence coding for thistetrapeptide. In lucerne and tobacco leaves, the levelof vicilin-KDEL protein was 20 and 100 times higherthan that of the unmodified vicilin, respectively(Wandelt et al., 1992). In the case of recombinantantibodies, it is very interesting that the recombinantfull-size antibodies do not accumulate in the cytosol,due to incorrect/incomplete assembly and folding ofheavy and light chain and consequent proteindegradation. Cytosolic accumulation of recombinantantibodies has only been successful for singlepolypeptide chains, such as antibody heavy or lightchains or scFvs (Schillberg et al., 2003).The protein-synthesis pathway is highly conservedbetween plants and animals, so human transgenes thatare expressed in plants yield proteins with identicalamino-acid sequences to their native counterparts.However, there are some important differences inpost-translational modification. The main differencebetween proteins that are produced in animals and82 Kunka Kamenarova et al.plants, however, concerns the synthesis of glycan sidechains. All eukaryotes add glycan chains to proteinsas they pass through the secretory pathway, but owingto differences in the levels of different modificationenzymes, the glycan-chain structures vary widelyacross different taxa (Ma et al., 2003). Plant-derivedrecombinant proteins tend to lack the terminalgalactose and sialic acid residues that are normallyfound in mammals, but have the carbohydrate groupα(1,3)-fucose, which has a (1,6) linkage in animalcells, and ß(1,2)-xylose, which is absent in mammalsalthough present in invertebrates (Ma et al., 2003).These minor differences in glycan structure couldpotentially change the activity, biodistribution andlongevity of recombinant proteins compared with thenative forms. The possibility of plant-specific glycansinducing allergic responses in humans has beenconsidered (Ma et al., 2003) and the finding thathuman serum contains antibodies that are reactiveagainst these residues has been interpreted as evidencethat theα(1,3)-fucose and ß(1,2)-xylose residuesmight lead to adverse reactions (Ma et al., 2003).However, carbohydrates are rarely allergenic.Moreover, the presence of antibodies in serum is notindicative of an adverse reaction. Finally, these glycanresidues are also associated with every normal plantglycoprotein that is found in our diet. So, it is highlyunlikely that they will be associated with adversereactions. Indeed, studies in which mice wereadministered a recombinant antibody that containedplant-specific glycans showed no evidence of anantiglycan immune reaction (Ma et al., 2003).Nevertheless, the perceived negative effect of‘foreign’ glycan structures is one of the mostimportant issues that affect the use and acceptance ofplant-derived recombinant proteins. Therefore, recentattention has focused on the development of strategiesto ‘humanize’ the glycosylation patterns ofrecombinant proteins. Strategies that have beenattempted in transgenic plants include the use ofpurified human ß(1,4)-galactosyltransferase andsialyltransferase enzymes to modify plant-derivedrecombinant proteins in vitro (Ma et al., 2003), and theexpression of human ß(1,4)-galactosyltransferase intransgenic tobacco plants to produce recombinantantibodies with galactose-extended glycans. In thelatter case, ~30% of the recovered antibody wasgalactosylated (Ma et al., 2003). In vivo sialylation isunlikely to be achieved in the near future becauseplants seem to lack the metabolic pathway for theprecursors of sialic acid, so several new enzymeswould need to be introduced and coordinatelyexpressed.Plant-expression hostsThe range of plant species amenable to transformationis growing at a phenomenal rate and it is unclear atpresent which species are optimal for molecularfarming. Many factors need to be taken intoconsideration (Schillberg et al., 2003). The yield offunctional protein in a given species needs to beevaluated carefully, since this factor has to be weighedagainst the total biomass yield over a given plantedarea and any associated overhead costs. The storageand distribution of the product is also a consideration.The costs of grain storage and distribution are minimalcompared with those of freshly-harvested tobaccoleaves or tomato fruits, but the costs of extraction andpurification are lower for watery plant material thandesiccated seed. The compromise between productioncosts and profit is likely to be a key factor in selectingthe crops used, because most pharmaceuticals will beproduced by industry.Ma end colleagues (Ma et al., 2003) have arrangedthe most spread plant production systems in threegroups: 1) tobacco production system; 2) cereals andlegumes and 3) fruit and vegetables.TobaccoTobacco has an established history as a routine systemfor molecular farming. The main advantages oftobacco include the mature technology for genetransfer and expression, the high biomass yield, thepotential for rapid scale-up owing to prolific seedproduction, and the availability of large-scaleinfrastructure for processing. Although many tobaccocultivars produce high levels of toxic alkaloids, thereare low-alkaloid varieties that can be used for theproduction of pharmaceutical proteins (Ma et al.,2003).As an alternative to nuclear transgenics,transplastomic plants are produced by introducingDNA into the chloroplast genome rather than thenuclear genome, a process that is generally achievedby particle bombardment. Human growth hormone,serum albumin, a tetanus toxin fragment and theMolecular farming in plants 83cholera toxin B subunit have been produced at highlevels in tobacco chloroplasts, and found to bestructurally authentic and biologically active. Thesedata show that plastids can fold and assembleoligomeric proteins correctly (Ma et al., 2003). Onedisadvantage of the chloroplast transgenic system isthat plastids do not carry out glycosylation. It istherefore unlikely that chloroplasts could be used tosynthesize human glycoproteins in cases in which theglycan-chain structure is crucial for protein activity.One of the disadvantages of recombinant-proteinproduction in tobacco is the instability of the product,which means that the leaf tissue must be frozen ordried for transport, or processed at the farm.Cereals and legumesThe accumulation of recombinant antibodies in seedsallows long-term storage at ambient temperaturesbecause the proteins amass in a stable form. Seedshave the appropriate biochemical environment forprotein accumulation, and achieve this through thecreation of specialized storage compartments, such asprotein bodies and storage vacuoles, which are derivedfrom the secretory pathway. Seeds are also desiccated,which reduces the exposure of stored proteins to non-enzymatic hydrolysis and protease degradation. Cerealseeds also lack the phenolic substances that are presentin tobacco leaves, so increasing the efficiency ofdownstream processing (Ma et al., 2003).Maize is now the main commercial productioncrop for recombinant proteins, which reflectsadvantages such as high biomass yield, ease oftransformation and in vitro manipulation, and ease ofscale-up. Maize is also being used for the productionof recombinant antibodies (Hodd et al., 2002a) andfurther technical/pharmaceutical enzymes, such aslaccase, trypsin and aprotinin (Hood, 2002b).The use of barley grains as bioreactors for highlyactive and thermo-tolerant hybrid cellulase (1,4-ß-glucanase) was investigated (Xue et al., 2003). Ofcrescent interest are the production of marker-freetransgenic plants and the use of cultivars withoutherbicide or antibiotic resistance. Toward this,transgenic barley plants whose genome contains genesfor production of human antithrombin III,α1-antitrypsin, lysozyme, serum albumin and lactoferrinwere generated (Stahl et al., 2002). Successfulexpression of human lactoferrin was achieved in riceby Anzai and colleagues (Anzai et al., 2000).Recombinant antibody of a single-chain Fv againstcarcinoembryonic antigen was produced in rice andwheat. It was confirmed that this antibody can bestored for at least five months at room temperature,without significant loss of the amount or the activity(Stöger et al., 2000).Alfalfa and soybean produce lower amounts of leafbiomass than tobacco, but have the advantage of usingatmospheric nitrogen through nitrogen fixation,thereby reducing the need for chemical inputs. Bothspecies have been used to produce recombinantantibodies (Ma et al., 2003). Pea is being developed asa production system, although at present the yields thatare possible with this species are low (Ma et al., 2003).Fruit and vegetablesThe main benefit of fruit, vegetable and leafy saladcrops is that they can be consumed raw or partiallyprocessed, which makes them particularly suitable forthe production of recombinant subunit vaccines, foodadditives and antibodies for topical passiveimmunotherapy (Ma et al., 2003). Potatoes have beenwidely used for the production of plant-derivedvaccines and have been administered to humans inmost of the clinical trials. The potential of potatotubers for antibody production was first shown byArtsaenko and colleagues (Artsaenko, 1998; Ma et al.,2003), and recently this crop has been investigated asa possible bulk-production system for antibodies (Maet al., 2003). Potatoes have also been used for theproduction of diagnostic antibody-fusion proteins andhuman milk proteins (Ma et al., 2003).Tomatoes,which were used to produce the first plant-derivedrabies vaccine (Ma et al., 2003), are more palatablethan potatoes and offer other advantages includinghigh biomass yields (~68,000 kg per hectare) and theincreased containment that is offered by growth ingreenhouses. Lettuce is also being investigated as aproduction host for edible recombinant vaccines, andhas been used in one series of clinical trials for avaccine against HBV (Ma et al., 2003). Bananas havebeen considered as hosts for the production ofrecombinant vaccines, as they are widely grown in thecountries in which vaccines are most needed and canbe consumed raw or as a puree by both adults andchildren (Ma et al., 2003).84 Kunka Kamenarova et al.Discussions and conclusionsLike many other aspects of crop biotechnology,supporters and critics of PMP crops differ stronglyover the benefits and risks of this new application.Proponents stress the societal benefits of a cheaper andmore plentiful source of pharmaceuticals, whileopponents emphasize the risks of contamination of thefood supply and unknown effects on ecosystems.Given the uncertainties surrounding bio-pharm crops,it is difficult to predict whether and to what extent thistechnology will become part of our future agriculturaland health care systems. Several questions remain tobe answered, including: (1) Are PMPs safe andeffective medicines for humans and animals? (2) Willproduction costs of PMPs, especially for thepurification process, be reduced sufficiently to bringthe promised economic benefits? (3) What will be theappropriate combinations of crop species, plant parts,growing environments, and production safeguards thatwill provide acceptable levels of gene containmentand environmental protection? (4) Are our regulatorystructures adequate to the task of regulating andmonitoring bio-pharm crops, and, if not, what changeswill be necessary? (5) To what extent will crop-basedpharmaceuticals provide new economic opportunitiesfor farmers and rural communities?Sales are a good measure of the public’s perceivedbenefits of specific products. However, today’s publicalso wants to know that not only is there a benefit forthe direct end user, but that there are otherwise nosignificant risks to the general public. This isillustrated by the recent concerns and debates over theuse of GMO products produced in plants. While theinitial concern involved GMO food products, this nowencompasses non-food products. The fear is that thenon-food products may inadvertently enter the foodchain and present an unintentional risk (Horn et al.,2004). The use of plants to produce non-food productsis not unlike the current use of other food products,such as eggs or yeast, which produce pharmaceuticals.The difference is the latter have well-establishedcompliance programs, which are in line with theproduction of pharmaceutical products rather than theproduction of food. Such compliance programs startedwith the arising of regulatory agencies that representthe public’s safety concerns. The regulatory agenciestake the position that the non-food products are unsafeuntil proven otherwise. There is a regulatoryframework in place specifically targeted toward theintroduction of non-food products when using plantsas the production system. There are strict rules onagronomic practices, which are targeted to keep non-food products out of the food chain. Unfortunately, inany system including plants it is not possible toeliminate all possibilities of unintended exposure dueto unforeseen circumstances such as an accident, anatural disaster, etc.One of the keys to success in the future willundoubtedly be the level of expression of therecombinant protein in plants. This is one of the mostimportant aspects with regard to economics.Expression is also a major regulatory concern (Horn etal., 2004). Whether or not the protein is in specifictissues will enable or nullify exposure to theenvironment. There has already been work to showthat expression can be limited to specific tissues, thusreducing regulatory concerns. As an example, keepingthe protein out of pollen can reduce inadvertentexposure to the environment. However, this does notremove the possibility that the pollen will outcrosswith other plants and intermix with food crops. Thereare some cases where genetic control of expression isalso warranted either for economic or safety concerns,depending on the product. Possibilities includingmale-sterile crops, induced expression, or sequencesthat prevent germination or the expression of theprotein product in non-food products have beendiscussed. Some combination of these differentlimitations on expression will most likely find a wayinto future programs.The other regulatory concern is that the pathway tocommercialization for human therapeutics has notbeen proven (Horn et al., 2004). With the firstapproved therapeutic products will also come therealization of the many benefits of transgenic planttechnology. These real benefits should also help publicacceptance and open the way for a much more rapidacceptance of this technology.ReferenceAnzai H, Takaiwa F and Katsumata K. Production of humanlactoferrin in transgenic plants. Elsevier Science B.V.265-271, 2000.Artsaenko O, Kettig B, Fiedler U, Conrad U. and Düring K.Potato tubers as a biofactory for recombinant antibodies.Mol. Breeding.4: 313-319, 1998.Barta A. The expression of nopaline synthase human growthMolecular farming in plants 85hormone chimaeric gene in transformed tobacco andsunflower callus tissue. Plant Mol. Biol. 6: 347-357,1986.During K. Wound-inducible expression and secretion of T4lysozyme and monoclonal antibodies in Nicotianatabacum. Ph. D Thesis. Mathematisch-Naturwissenschaftlichen Fakultat der Universität zuKöln, 1988.Freese B. Manufacturing drugs and chemical crops:Biopharming poses new threats to consumers, farmers,food companies and the environment. Available fromGE Food Alert, www.gefoodalert.org, 2002.Hiatt A, Cafferkey R and Bowdish K. Production ofantibodies in transgenic plants, Nature. 342 (6245): 76-78, 1989.Hood EE, Kusnadi A, Nikolov Z, Howard JA. Molecularfarming of industrial proteins from transgenic maize. In:Chemicals via higher plant bioengineering.Kluwer/Plenum.Shahidi F, Kolodziejczyk P, WhitakerJR, Munguia AL, Fuller G, New York, 127–147, 1999.Hood EE, Witcher DR, Maddock S, Meyer T, BaszczynskiC, Bailey M, Flynn P, Register J, Marshall L, Bond D,Kulisek E, Kusnadi A, Evangelista R, Nikolov Z, WoogeC, Mehigh RJ, Hernan R, Kappel WK, Ritland D, Li CPand Howard JA. Commercial production of avidin fromtransgenic maize: characterization of transformant,production, processing, extraction and purification. MolBreed 3: 291–306, 1997.Hood EE. From green plants to industrial enzymes. EnzymeMicrobial Technol. 30: 279–283, 2002a.Hood EE, Woodard SL and Horn ME. Monoclonal antibodymanufacturing in transgenic plants - myths and realities.Curr. Opin. Biotechnol. 13: 630–635, 2002b.Horn ME, Woodard SL and Howard JA. Plant molecularfarming: systems and products. Plant Cell Rep. 22:711–720, 2004.Kusnadi AR, Hood EE, Witcher DR, Howard JA andNikolov ZL. Production and purification of tworecombinant proteins from transgenic corn. BiotechnolProg. 14: 149–155, 1998.Lamphear BJ, Jilka JM, Kesl L, Welter M, Howard JA andStreatfield SJ. A corn-based delivery system for animalvaccines: an oral transmissable gastroenteritis virusvaccine boosts lactogenic immunity in swine. Vaccine(in press), 2004.Ma JK-C, Drake PMW and Christou P. The production ofrecombinant pharmaceutical proteins in plants. Genetics.4: 794-805, 2003.Schillberg S, Fischer R and Emans N. Molecular farming ofantibodies in plants. Naturwissenschften. 90: 145-155,2003.Seon J-H, Szarka JS and Moloney MM. A unique strategyfor recovering recombinant proteins from molecularfarming: affinity capture on engineered oilbodies. J.Plant Biotechnology.4 (3): 95-101, 2002.Stahl R, Horvath H, Van Fleet J, Voetz M, Von Wettstein Dand Wolf N. T-DNA integration into the barley genomefrom single and double cassette vectors. Proc. Natl.Acad. Sci. 99 (4): 2146-2151, 2002.Stöger E, Vaquero C, Torres E, Sack M, Nicholson L,Drossard J, Williams S, Keen D, Perrin Y, Christou PandFischer R. Cereal crops as viable production and storagesystems for pharmaceutical scFV antibodies. PlantMol.Biol.42: 583-590, 2000.Suslow TV, Thomas BR and Bradford KJ. Biotechnologyprovides new tools for planting. Oakland: University ofCalifornia Division of Agriculture and NaturalResources, Publication 8043, 2002.Thomas BR, Van Deynze Aand Bradford KJ. Production ofTherapeutic proteins in plants. AgriculturalBiotechnology in California Series, Publication 8078,2002.Wandelt CI, Khan MR, Craig S, Schroeder HE, Spencer Danf Higgins TJ. Vicilin with carboxy-terminal KDEL isretained in the endoplasmic reticulum and accumulatesto high levels in the leaves of transgenic plants. ThePlant Journal. 2: 181, 1992.Xue GP, Patel M, Johnson JC and Smith DJ. Selectablemarker-free transgenic barley producing a high-level ofcellulase (1,4-ß-glucanase) in developing grains. PlantCell Rep. 21: 1088-1094, 2003.Zhong G-Y, Peterson D, Delaney DE, Bailey M, WitcherDR, Register JC III, Bond D, Li C-P, Marshall L,Kulisek E, Ritland D, Meyer T, Hood EE and HowardJA. Commercial production of aprotinin in transgenicmaize seeds. Mol Breed. 5: 345–356, 1999.86 Kunka Kamenarova et al.